CN108379655B - Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof - Google Patents
Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof Download PDFInfo
- Publication number
- CN108379655B CN108379655B CN201810497604.XA CN201810497604A CN108379655B CN 108379655 B CN108379655 B CN 108379655B CN 201810497604 A CN201810497604 A CN 201810497604A CN 108379655 B CN108379655 B CN 108379655B
- Authority
- CN
- China
- Prior art keywords
- nerve
- gel solution
- nerve graft
- solution
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000005036 nerve Anatomy 0.000 title claims abstract description 82
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 63
- 239000000835 fiber Substances 0.000 claims abstract description 40
- 238000010041 electrostatic spinning Methods 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229920000642 polymer Polymers 0.000 claims abstract description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000007704 transition Effects 0.000 claims abstract description 5
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims abstract description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 86
- 239000000499 gel Substances 0.000 claims description 52
- 238000000034 method Methods 0.000 claims description 30
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 24
- 102000007072 Nerve Growth Factors Human genes 0.000 claims description 24
- 239000003900 neurotrophic factor Substances 0.000 claims description 24
- 239000011159 matrix material Substances 0.000 claims description 22
- 210000000578 peripheral nerve Anatomy 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 229920001410 Microfiber Polymers 0.000 claims description 11
- 239000003658 microfiber Substances 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 claims description 9
- 238000002791 soaking Methods 0.000 claims description 9
- 238000001523 electrospinning Methods 0.000 claims description 7
- 238000001879 gelation Methods 0.000 claims description 7
- 210000002950 fibroblast Anatomy 0.000 claims description 4
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 3
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 229920000954 Polyglycolide Polymers 0.000 claims description 3
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 3
- 239000004626 polylactic acid Substances 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 2
- 108010035532 Collagen Proteins 0.000 claims description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 claims description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 2
- 229920001436 collagen Polymers 0.000 claims description 2
- 229920001610 polycaprolactone Polymers 0.000 claims description 2
- 239000004632 polycaprolactone Substances 0.000 claims description 2
- 239000004633 polyglycolic acid Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 1
- 230000008929 regeneration Effects 0.000 abstract description 10
- 238000011069 regeneration method Methods 0.000 abstract description 10
- 239000000017 hydrogel Substances 0.000 description 15
- 238000009987 spinning Methods 0.000 description 13
- 239000000463 material Substances 0.000 description 12
- 230000005684 electric field Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 238000004108 freeze drying Methods 0.000 description 7
- 210000002569 neuron Anatomy 0.000 description 7
- 230000009471 action Effects 0.000 description 6
- 239000002657 fibrous material Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 5
- 210000003050 axon Anatomy 0.000 description 5
- 230000007547 defect Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 229920001432 poly(L-lactide) Polymers 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 208000010886 Peripheral nerve injury Diseases 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 239000000645 desinfectant Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000009434 installation Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000001079 digestive effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 229960003942 amphotericin b Drugs 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000028600 axonogenesis Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000002121 nanofiber Substances 0.000 description 2
- 230000000926 neurological effect Effects 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 239000008279 sol Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008093 supporting effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- 230000001228 trophic effect Effects 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010039203 Road traffic accident Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 230000000482 effect on migration Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000005709 nerve cell growth Effects 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
- D01D5/0038—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion the fibre formed by solvent evaporation, i.e. dry electro-spinning
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/12—Stretch-spinning methods
- D01D5/14—Stretch-spinning methods with flowing liquid or gaseous stretching media, e.g. solution-blowing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Transplantation (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Textile Engineering (AREA)
- Mechanical Engineering (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Vascular Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Neurosurgery (AREA)
- Biophysics (AREA)
- Neurology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to a nerve graft with a three-dimensional orientation structure, a preparation method and manufacturing equipment thereof, and relates to the technical field of tissue engineering. The preparation method of the nerve graft with the three-dimensional orientation structure comprises the following steps: the polymer solution is subjected to electrostatic spinning to form fibers and falls into receiving liquid, wherein the receiving liquid directionally flows to be in a laminar flow or transition flow state; the receiving liquid is selected from any one of cyclohexane and C1-C6 alcohol, preferably methanol, ethanol or propanol. It can provide better mechanical support and physical cell guiding function, and effectively guide nerve regeneration. The preparation method is controllable, convenient and reliable in operation, the preparation equipment for preparing the nerve graft with the three-dimensional orientation structure is simple in structure, and meanwhile, the efficiency of preparing the nerve graft with the three-dimensional orientation structure is effectively improved.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a nerve graft with a three-dimensional orientation structure, and a preparation method and manufacturing equipment thereof.
Background
Along with the development of social economy, the mechanization degree is increasingly improved, the population mobility is high, and peripheral nerve injuries can be caused by traffic accidents, sports accidents and the like. The self-repair ability of peripheral nerves of human is limited, peripheral nerve injury is always permanent, and repair and functional reconstruction after peripheral nerve injury are always a great problem in clinical medical research. Clinical treatment of short-range neurological defects can be achieved by suturing end-to-end, but for medium-and long-range neurological defects a nerve graft is required to bridge repair. Currently, the standard for repair of peripheral nerve injury is autologous nerve transplantation.
The autologous nerve transplantation can provide sufficient cells which are beneficial to the regeneration of the axon of the nerve at the injury site, and the radial channel in the autologous nerve cavity also provides a good physical guide function for the regeneration of the axon. However, there are many limitations to the use of autologous nerve transplantation for treatment, such as: the source of nerve graft is less, the donor part is diseased, the size of the donor nerve is not consistent, and a plurality of operations are needed. (Nerve guide details Based on Double-Layered scaffold of electrospun nanofibrers for repaying the Peripheral Nerve System) therefore, Based on the principle of tissue engineering, it is necessary and meaningful to develop artificial Nerve graft instead of autograft, and a new strategy can be provided for the treatment of Peripheral Nerve defects.
An ideal nerve bridge should have the following characteristics: (1) good biocompatibility and in vivo degradation performance; (2) the material has plasticity, adjustable size and certain mechanical strength; (3) has physical guiding effect on nerve migration and axon regeneration; (4) providing trophic factors needed by nerve regeneration, regulating the growth and differentiation of nerve cells, and increasing the growth speed of axons.
Disclosure of Invention
The invention aims to provide a nerve graft with a three-dimensional orientation structure, which can provide better mechanical support and physical cell guiding effect and effectively guide nerve regeneration.
The invention also aims to provide a preparation method of the nerve graft with the three-dimensional orientation structure, which has controllable, convenient and reliable operation.
Another object of the present invention is to provide a manufacturing apparatus for preparing a nerve graft having a three-dimensional oriented structure, which has a simple structure and effectively improves the efficiency of preparing a nerve graft having a three-dimensional oriented structure.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a preparation method of a nerve graft with a three-dimensional orientation structure, which comprises the following steps:
and (3) forming fibers by electrostatic spinning of the polymer solution and dropping the fibers into a receiving liquid, wherein the receiving liquid directionally flows to be in a laminar flow or transition flow state.
The receiving liquid is selected from any one of cyclohexane and C1-C6 alcohol, preferably methanol, ethanol or propanol.
The invention provides a nerve graft with a three-dimensional orientation structure, which is prepared by the preparation method.
The invention provides a manufacturing device for preparing the nerve graft with the three-dimensional orientation structure, which comprises electrostatic spinning equipment and rotary receiving equipment, wherein the rotary receiving equipment comprises a base, a motor and a rotary table, the base is detachably connected with the rotary table, the base is provided with an installation groove, the motor is arranged in the installation groove and is coaxially connected with the rotary table for driving the rotary table to rotate, and the motor is provided with a speed controller.
One side of the turntable, which is far away from the base, is provided with an accommodating cavity, the accommodating cavity is provided with a fixing shaft protruding out of the bottom wall of the accommodating cavity, the fixing shaft is a cylinder, an annular liquid tank is formed between the fixing shaft and the accommodating cavity, the electrostatic spinning equipment comprises an injection needle, and the injection needle is positioned above the liquid tank and is arranged at an interval with the liquid tank.
The nerve graft with the three-dimensional orientation structure, the preparation method and the manufacturing equipment thereof have the advantages that:
high voltage is applied to polymer solution (spinning solution), the polymer solution with the same charge repels each other to form small droplet solvent which is continuously volatilized to form jet flow, and simultaneously, the fiber is quickly formed to be sprayed to a receiving plate (a low electric field position) under the stretching action of electric field force. The fiber is gradually solidified along with the evaporation of the solvent in the air flight process, and finally falls into the receiving liquid which is in a laminar or transitional flow state and flows directionally in the rotary receiving equipment. The fiber is in a suspension state in the receiving liquid and directionally flows along the direction of the liquid flow, the liquid flow has a stretching orientation effect on the fiber, and the middle part of the rotary receiving device is a cylindrical fixed shaft, so that the stable directional flow of the liquid in the liquid tank can not form vortex during rotation, and finally the fiber forms a circle to form a fiber bundle with a certain diameter to form a three-dimensional orientation structure.
Meanwhile, the rotary liquid receiving operation is simple, and the oriented electrostatic spinning fiber material with a three-dimensional structure can be efficiently prepared. The prepared oriented micron fiber can provide physical guide effect on migration and growth of nerve cells and blood cells. Meanwhile, the three-dimensional oriented fiber material prepared by the synthetic polymer has high mechanical strength and good supporting effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
Fig. 1 is a schematic structural diagram of a rotary receiving apparatus provided in embodiment 1 of the present invention;
FIG. 2 is a schematic diagram of a nerve graft soaked in pure water for use according to example 2 of the present invention;
fig. 3A is a pictorial view of a lyophilized nerve graft provided in example 2 of the present invention;
FIG. 3B is a scanning electron microscope microstructure of the nerve graft of FIG. 3;
FIG. 4 is a pictorial representation of a pDNM pre-gel solution provided in example 3 of the present invention;
FIG. 5 is a schematic diagram of pDNM-gel provided in example 3 of the present invention;
FIG. 6 is a microscopic view of pDNM-gel provided in example 3 of the present invention;
fig. 7A is a pictorial view of a nerve graft provided in example 3 of the present invention before lyophilization;
fig. 7B is a pictorial view of a nerve graft provided in example 3 of the present invention before lyophilization;
fig. 8 is a scanning electron micrograph of a lyophilized nerve graft provided in example 3 of the present invention.
Icon: 10-a rotating receiving device; 11-a base; 111-mounting grooves; 12-a motor; 13-a turntable; 131-a containment chamber; 133-a fixed shaft; 135-liquid bath.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The nerve graft having a three-dimensional orientation structure according to the embodiment of the present invention, and a method and an apparatus for manufacturing the same will be described in detail below.
Because axons grow better on oriented fibers than on randomly oriented fiber scaffolds, oriented fibers are more conducive to the elongation of neural stem cells and axon growth. Meanwhile, the diameter of the fiber can also influence the differentiation of nerve cells, and the nanofiber is more beneficial to the differentiation of nerve stem cells.
Accordingly, the present invention provides a nerve graft having a three-dimensional oriented structure, which is prepared by the following method:
s1, forming fibers by electrostatic spinning of a polymer solution and enabling the fibers to fall into receiving liquid, wherein the receiving liquid directionally flows to be in a laminar flow or transition flow state, and thus the micron fibers with three-dimensional orientation structures are obtained.
The action mechanism is as follows: high voltage is applied to polymer solution (spinning solution), the polymer solution with the same charge repels each other to form small droplet solvent which is continuously volatilized to form jet flow, and simultaneously, the fiber is quickly formed to be sprayed to a receiving plate (a low electric field position) under the stretching action of electric field force. The fiber is gradually solidified along with the evaporation of the solvent in the air flight process, and finally falls into the receiving liquid which directionally flows to be in a laminar flow or transition flow state. The fiber is in a suspension state in the receiving liquid and flows directionally along with the direction of the liquid flow, the liquid flow has a stretching orientation effect on the fiber, meanwhile, the directional flow does not form a vortex, and finally, the fiber forms a circle to form a fiber bundle with a certain diameter.
It should be noted that the receiving liquid needs to flow directionally, so as to effectively avoid forming vortex, form laminar flow or transitional flow state, and make the final fiber form a circle to form a fiber bundle with a certain diameter, forming a three-dimensional orientation structure.
In the preferred embodiment of the present invention, the receiving liquid is processed at 40-60 r/min. Stirring rates of, for example, 43r/min, 45r/min, 46r/min, 48r/min, 53r/min or 55r/min to form laminar or transitional flow conditions; the laminar or transitional flow regime created allows for the optimal structure of the resulting microfibers with three-dimensional orientation.
Further, in a preferred embodiment of the present invention, the receiving liquid is selected from any one of cyclohexane and C1-C6 alcohol. Preferably methanol, ethanol or propanol, which on the one hand is convenient for subsequent removal and is simultaneously non-toxic and on the other hand is convenient for access.
In a preferred embodiment of the present invention, the polymer is selected from any one of levorotatory polylactic acid, polycaprolactone, polyglycolic acid, and polyglycolide; the biodegradable plastic has good biocompatibility and is degradable, and better mechanical support is provided. The polymer solution is obtained by mixing a polymer with a solvent, and is used as a spinning solution. Among them, the solvent is a reagent which is non-toxic and has better solubility for the polymer, such as trifluoroethanol, etc., and the prior art can be referred to herein.
Preferably, the polymer solution is ultrasonically oscillated before electrostatic spinning to remove air in the polymer solution, so that the quality of the micron fiber with the three-dimensional orientation structure obtained subsequently is uniform.
The invention adopts the electrostatic spinning technology to effectively obtain micron and/or nano fibers, the electrostatic spinning is a special fiber manufacturing process, polymer solution is subjected to jet spinning in a strong electric field, liquid drops at a needle head are changed into a cone shape (a Taylor cone) from a sphere under the action of the electric field, and fiber filaments are obtained by extending from the tip of the cone, and the polymer filaments with the nanometer-scale diameter can be produced by the method.
In a preferred embodiment of the present invention, the injection speed of the spinning solution in the electrostatic spinning is 1-4 mL/h, such as 1-3 mL/h, 2-4 mL/h or 1.5-3.5 mL/h. Preferably, the distance between the tip of the electrostatic spinning needle and the liquid surface of the receiving liquid is 4-10 cm, such as 4cm, 5cm, 6cm, 7.5cm, 8.5cm or 9cm, and the needle is connected with high voltage of +10kV to +15 kV. Under the conditions, the electrostatic spinning effect is good.
In the preferred embodiment of the invention, the method further comprises removing the receiving liquid on the surface of the microfiber; prevent the receiving liquid from influencing the activity of subsequent nerve cells or causing certain side effect on the nerve cells.
Preferably, in a preferred embodiment of the present invention, the receiving liquid for removing the surface of the microfiber comprises: washing with ethanol, soaking in pure water, and sucking water attached to the surface of the micrometer fibers.
In a preferred embodiment of the invention, the method further comprises the steps of precooling for 1-2 hours at-30 to-50 ℃ after the receiving liquid is removed, freezing water to keep the shape and structure of the support material, and then freeze-drying to obtain a dried framework, which is convenient for subsequent scientific research, storage, transportation and the like. Meanwhile, the precooling and freezing modes are adopted, so that the shrinkage rate of the powder in the drying process is inhibited while the powder is effectively dried, and the subsequent operation is more accurately performed. For example-45 deg.C, -47 deg.C or-50 deg.C, and vacuum is applied in a frozen state to sublimate ice in the material to obtain a dried material. Compared with normal-temperature drying, freeze-drying can better maintain the structure of the material and prevent the material from collapsing.
The obtained micrometer fibers with the three-dimensional orientation structure can be used as a nerve graft to provide better mechanical support and physical cell guiding functions, and effectively guide nerve regeneration. However, an ideal nerve bridge should have the trophic factors needed to provide nerve regeneration, regulate nerve cell growth, differentiation, and increase the axon growth rate. Meanwhile, the single physical guide function, the bridging bracket lacking biological activity and biological nutrient substances is not beneficial to the repair of long-section nerves.
Therefore, in a preferred embodiment of the present invention, the method further comprises:
s2, soaking the micron fibers with the three-dimensional orientation structures in a pre-gelling solution for gelling. The problems are effectively solved. The pre-gel solution is a gel solution that is in a solution state before the hydrogel is not gelled, and the pre-gel solution is in a hydrogel state after gelation.
The hydrogel is introduced and combined with the micro-fibers with three-dimensional oriented structures to form a micro-nano multilevel structure, the multilevel structure promotes the differentiation of nerve cells, and meanwhile, the acellular matrix hydrogel and the biological gel have good biological activity, improve the biocompatibility of materials and the like. At the moment, the three-dimensional oriented fiber material prepared by the synthetic polymer has high mechanical strength and can make up the defect of weak hydrogel strength when being used as a framework material in a nerve graft.
Wherein the pre-gel solution is in a cell matrix removing pre-gel solution or a biological pre-gel solution, and the biological pre-gel solution is selected from any one of a pre-gel solution of gelatin, a pre-gel solution of collagen and a polypeptide pre-gel solution. For example, the biological pre-gel solution is a RADA polypeptide pre-gel solution or a gelatin pre-gel solution. In a preferred embodiment of the present invention, the cell matrix-removed pre-gel solution is prepared by decellularizing and preparing a cell matrix-removed pre-gel solution from tissue of a large-scale animal such as pig, cow or sheep. Meanwhile, the acellular matrix pre-gel solution comprises tissues such as brain, spinal cord, small intestine submucosa or pericardium.
Preferably, in a preferred embodiment of the present invention, the acellular matrix pre-gel solution is a neural acellular matrix pre-gel solution; preferably, the acellular matrix pre-gel solution is preferably porcine-derived peripheral nerve acellular matrix pre-gel solution (pDNM-gel). The introduction of pDNM-gel is combined with the oriented electrostatic spinning fiber material with a three-dimensional structure to form a micro-nano multistage oriented structure, so that the differentiation of nerve cells is effectively promoted, and meanwhile, the pDNM-gel has good biological activity, the biocompatibility is further improved, and the like.
The acellular matrix hydrogel may be purchased directly from the market or prepared according to the existing method, and those skilled in the art may make limitations according to actual needs, and is not specifically limited herein.
Preferably, the microfiber with a three-dimensional orientation structure is soaked in a pre-gel solution at a temperature of less than 37 ℃, preferably 2 to 10 ℃, for example, 3, 4, 5 or 6 ℃, and then is gelled at 37 ℃. Here, the gelation at 37 ℃ means gelation of the pre-gel solution, that is, the temperature of curing is 37 ℃. The solution is in a sol state at the temperature of less than 37 ℃, so that the soaking efficiency and the soaking sufficiency are effectively improved.
Optionally, after gelation, scraping off excess hydrogel on the surface of the obtained material.
In the preferred embodiment of the invention, the neurotrophic factors can be added according to the requirements, which is beneficial to the regeneration of long-section nerve defects.
In a preferred embodiment of the present invention, the method further comprises adding a neurotrophic factor to the pre-gel solution before gelation; at the moment, the nerve nutrition factor and the solution-like pre-gel solution are effectively and uniformly mixed and loaded on the micrometer fibers with the three-dimensional orientation structure, so that the quality uniformity of the nerve graft is improved.
In a preferred embodiment of the present invention, the concentration of neurotrophic factor in the pre-gel solution is preferably 1ug/mL to 20mg/mL, such as 10ug/mL, 30ug/mL, 60ug/mL, 10mg/mL, 40mg/mL, 6mg/mL, 8mg/mL, 10mg/mL, 14mg/mL, 17mg/mL, 19mg/mL, etc.
In a preferred embodiment of the present invention, the neurotrophic factor preferably comprises at least one of a neurotrophic factor, a basic fibroblast neurotrophic factor, a brain-derived neurotrophic factor, a neurotrophic factor 3, and a glial cell-derived neurotrophic factor. For example, the neurotrophic factor is a neurotrophic factor or a basic fibroblast neurotrophic factor, or a mixture of a brain-derived neurotrophic factor and neurotrophic factor 3.
The invention also provides a manufacturing device for preparing the nerve graft with the three-dimensional orientation structure, which comprises electrostatic spinning equipment and rotary receiving equipment, wherein the rotary receiving equipment comprises a base, a motor and a turntable, the base is detachably connected with the turntable, the base is provided with an installation groove, the motor is arranged in the installation groove, the motor is coaxially connected with the turntable and used for driving the turntable to rotate, and the motor is provided with a speed controller.
Specifically, one side of the turntable, which is far away from the base, is provided with an accommodating cavity, the accommodating cavity is provided with a fixing shaft protruding out of the bottom wall of the accommodating cavity, the fixing shaft is a cylinder, an annular liquid tank is formed between the fixing shaft and the accommodating cavity, the electrostatic spinning equipment comprises an injection needle, and the injection needle is positioned above the liquid tank and is arranged at an interval with the liquid tank.
Preferably, the accommodating cavity is a cylindrical accommodating cavity, and the diameter of the fixing shaft is 1/4-2/5 of the diameter of the accommodating cavity. The manufacturing equipment is simple in structure, and meanwhile, the efficiency of preparing the nerve graft with the three-dimensional orientation structure is effectively improved.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Referring to fig. 1, the present embodiment provides a manufacturing apparatus (not shown) for preparing a nerve graft having a three-dimensional orientation structure, which includes an electrospinning apparatus (not shown) and a rotary receiving apparatus 10.
The electrostatic spinning equipment is the existing equipment, and is not described in detail herein. The electrospinning device includes an injector (not shown) having an injection needle for performing jet spinning of the polymer solution at the needle in a strong electric field under the action of the electric field. Under the action of the electric field, the liquid drop at the needle head changes from a spherical shape to a conical shape (i.e. a Taylor cone), and a polymer filament with the diameter of a micron or nanometer scale is obtained by extending from the tip of the cone.
The rotary receiving device 10 includes a base 11, a motor 12, and a rotary disk 13, wherein the base 11 is detachably connected to the rotary disk 13, and in this embodiment, the base 11 is preferably in a snap-fit connection with the rotary disk 13.
The base 11 is provided with a mounting groove 111, the motor 12 is disposed in the mounting groove 111, an output shaft of the motor 12 is coaxially connected with the rotary table 13 for driving the rotary table 13 and the motor 12 to synchronously rotate, and the motor 12 is provided with a speed controller (not shown).
The side of the rotary disk 13 far from the base 11 is provided with an accommodating cavity 131, the accommodating cavity 131 is provided with a fixed shaft 133 protruding out of the bottom wall of the accommodating cavity, an annular liquid groove 135 is formed between the fixed shaft 133 and the accommodating cavity 131, and the injection needle is positioned above the liquid groove 135 and is arranged at an interval with the liquid groove 135.
The fixing shaft 133 is a cylinder, the accommodating cavity 131 is a cylindrical accommodating cavity 131, the diameter of the fixing shaft 133 is 1/4-2/5 of the diameter of the accommodating cavity 131, and in the embodiment, the diameter of the fixing shaft 133 is 1/3 of the diameter of the accommodating cavity 131. Alternatively, the fixing shaft 133 may be hollow or solid, and is not limited herein. The axis of the fixed shaft 133 coincides with the axis of the accommodating chamber 131, that is, the fixed shaft 133 is located at the center of the accommodating chamber 131, so that the liquid in the liquid tank 135 flows more stably during the rotation. That is, the liquid tank 135 has an annular cross section.
Example 2
A nerve graft having a three-dimensional orientation structure, which is prepared by the following method using the manufacturing apparatus provided in example 1:
polylactic acid with the intrinsic viscosity of 3.8 is used as an effective component, trifluoroethanol is used as a solvent, a PLLA solution with the concentration of 10% (0.1g/mL) is prepared, and the PLLA solution is stirred and dissolved for 24 hours at normal temperature to prepare a polymer solution, namely a spinning solution.
Before electrostatic spinning, bubbles in the spinning solution are removed by an ultrasonic oscillation method, and then the spinning solution is added into an injector in electrostatic spinning equipment, and a national standard No. 9 plain-end needle head is used.
Before the electrostatic spinning device is started, adding ethanol into the liquid tank to serve as receiving liquid, wherein the adding amount of the ethanol is half of the depth of the liquid tank. Then the rotary receiving equipment is started, the motor drives the turntable to rotate, the rotating speed is about 1 circle/second, and therefore the receiving liquid in the liquid tank rotates to form laminar flow or transitional flow.
The electrostatic spinning injector is arranged on the upper part of the liquid in the liquid tank, the distance between the needle point and the liquid level is about 4cm, the needle head is connected with high voltage, the voltage is +10kV to +15kV, and the rotary receiving equipment is grounded. The injection speed of the spinning solution was 1 mL/h.
In the spinning process, the spinning solution is sprayed out to form a Taylor cone, the fiber falls into a liquid tank to be solidified after being formed, and simultaneously the fiber is uniformly suspended and dispersed in an ethanol solution and forms an oriented structure along with liquid flow.
After the electrostatic spinning is finished, the electrostatic spinning fibers in the liquid tank are taken out in strips, washed by 75 percent, 50 percent and 25 percent gradient ethanol for 2 hours respectively, and finally soaked in pure water for standby application, and the real object diagram of the nerve graft is shown in figure 2.
The size (length, diameter) of the electrostatic spinning can be controlled by controlling the amount of the spinning solution and the later cutting according to actual needs.
Precooling the nerve graft soaked in pure water for standby at-40 ℃ for 1 hour, and freeze-drying the nerve graft for 48 hours by using a freeze dryer to obtain a dried nerve graft, wherein the physical picture of the dried nerve graft is shown as figure 3A, and the scanning electron microscope picture of the dried nerve graft is shown as figure 3B.
As shown in fig. 3b, it was confirmed that the resulting nerve graft had an oriented structure and the diameter of the individual fiber was about 1 um.
Example 3
The present invention provides a nerve graft having a three-dimensional oriented structure obtained by soaking a nerve graft to be used in pure water as provided in example 2 in the form of a hydrogel supporting acellular matrix.
Specifically, in this example, the acellular matrix hydrogel is porcine-derived peripheral nerve acellular matrix hydrogel (pDNM-gel), and it should be noted that: the porcine peripheral nerve acellular matrix is named as pDNM for short, and the porcine-derived peripheral nerve acellular matrix hydrogel is named as pDNM-gel for short.
The porcine-derived peripheral nerve acellular matrix hydrogel is prepared by the following method:
1) preparation of pDNM:
the peripheral nerves of the pigs were obtained from the extremities of male long white pigs, and the surface adipose tissue and part of the adventitia were subtracted under an operating microscope. According to the whole preparation aseptic principle, the materials are taken and immediately put into the pre-cooled disinfectant. Preparing a disinfectant: sterile PBS + 0.1% V/V peroxyacetic acid + 4% V/V ethanol, filtering with 0.22 μm pore size filter, transferring into sterile culture medium bottle, and tissue volume is no more than 1/3 of disinfectant volume. The peripheral nerve tissue was rinsed 2 times with sterile PBS for 10 minutes each time to remove residual disinfectant components. The treated peripheral nerves were soaked in sterile PBS containing 2% double antibody (penicillin-streptomycin), 10. mu.g/mL gentamicin (gentamicin), 2.5. mu.g/mL amphotericin B (amphotericin B), and the next treatment was performed after 4 hours. The disinfected and sterilized nerve tissues are put in distilled water to be vibrated and rinsed for 6 hours. Then, the cell removing process is completed by a chemical extraction method: the nerves were put into a 3% TritonX100 aqueous solution and shaken for 12h (room temperature, 25 ℃), rinsed 3 times (room temperature, 25 ℃) in sterile distilled water; then put into 4% sodium deoxycholate aqueous solution and shake for 24h (room temperature, 25 ℃), and finally rinsed in sterile distilled water for 3 times (room temperature, 25 ℃), so that one cycle is extraction 1 time, and 2 cycles are performed in total. Freeze-drying peripheral nerve after extraction, and then using VEthanol/VMethylene dichlorideThe degreasing was performed with a mixed solvent of 1/2, and the degreased product was washed with sterile distilled water several times (to remove the residual organic solvent). The two lyophilizates were crushed using a mini mill (WileyWill), received through a 40 mesh screen, sealed and stored at 4 ℃ until ready for use, and the resulting pregel solution of pDMM was as shown in FIG. 4.
2) preparation of pDNM-gel:
a certain amount of pDMM powder is weighed, placed in hydrochloric acid solution of pepsin and digested for 24 h. Wherein the mass ratio of pDMM powder to pepsin is 10:1, and the hydrochloric acid concentration is properly adjusted according to the concentration of pDMM. After a certain time of digestion of pDMN, the undigested particles (mass fraction less than 5%) are removed by ultracentrifugation (rotating speed is properly increased according to the concentration of pDMN), and the obtained digestive juice is frozen at-40 ℃ for standby.
3) The preparation method of the gel comprises the steps of firstly utilizing NaOH solution to adjust the pH value of pDMM digestive juice to be more than or equal to 8 at 4 ℃, then adjusting the pH value back to 7.4, then introducing 1/9 10 × PBS with the volume of the digestive juice to obtain pDMM sol (as shown in figure 4), heating the pDMM pre-gel solution to 37 ℃ to generate sol-gelation transformation, keeping the temperature at 37 ℃ for 20 minutes to obtain pDMM-gel shown in figures 5 and 6, wherein the preparation processes are operated in a sterile environment, all the used solvents are filtered through a 0.22 mu m pore size filter in the sterile environment, and the solution-type porcine-derived peripheral nerve acellular matrix hydrogel is prepared.
The method for compounding the pDNM-gel and the PLLA three-dimensional oriented microfiber material comprises the following steps:
taking out the PLLA three-dimensional orientation micron fiber material soaked in pure water by using a pair of tweezers, sucking water adsorbed on the surface by using sterile filter paper, placing the filter paper in a culture dish, and adding a pDNNM pre-gel solution for soaking so as to completely soak the PLLA three-dimensional orientation micron fiber material in the pre-gel solution. And (4) putting the whole into a refrigerator at 4 ℃ for soaking for 12 hours at low temperature, transferring the whole into a thermostat at 37 ℃ for 20 minutes, and forming the gel by pDMN-gel. Scraping excess pDNM-gel from the surface of the material to obtain the final nerve graft, as shown in fig. 7.
The nerve graft shown in fig. 7A was pre-cooled at-40 degrees celsius for 1 hour, and after freeze-drying with a freeze dryer for 48 hours, a dried nerve graft was obtained as shown in fig. 7B.
The nerve graft obtained in fig. 7A is soaked and fixed with 2% glutaraldehyde for 2 hours, then washed and freeze-dried, and then is characterized by a scanning electron microscope, and the microstructure diagram is shown in fig. 8, which proves that the nerve graft has a micro-nano multilevel fiber structure.
Example 4
It differs from the nerve graft provided in example 3 in that: the porcine-derived peripheral nerve acellular matrix hydrogel further comprises neurotrophic factors, wherein the neurotrophic factors comprise: the nerve neurotrophic factor and the basic fibroblast neurotrophic factor, wherein the content of the neurotrophic factor added into the pDNM pre-gel solution is 1 mg/mL.
Wherein, the neurotrophic factor is added before pDNM-gel gelatinizing, namely, in the preparation method of the porcine-derived peripheral nerve acellular matrix hydrogel, the neurotrophic factor is added into a pre-gel solution before the temperature is raised to 37 ℃.
In summary, the embodiment of the present invention provides a nerve graft with a three-dimensional orientation structure, which can provide better mechanical support and physical cell guiding effects, and effectively guide nerve regeneration. The preparation method is controllable, convenient and reliable in operation. The manufacturing equipment for preparing the nerve graft with the three-dimensional orientation structure has a simple structure, and simultaneously, the efficiency of preparing the nerve graft with the three-dimensional orientation structure is effectively improved.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (19)
1. A method of preparing a nerve graft having a three-dimensional orientation structure, comprising:
forming fibers by electrostatic spinning of a polymer solution and enabling the fibers to fall into a receiving liquid, wherein the receiving liquid directionally flows in an annular liquid tank at a stirring speed of 40-60 r/min and is in a laminar flow or transition flow state;
the receiving liquid is selected from any one of cyclohexane and C1-C6 alcohol.
2. The method of claim 1, wherein the receiving solution is methanol, ethanol, or propanol.
3. The method according to claim 1, wherein the polymer solution is injected at a rate of 1 to 4mL/h in the electrospinning.
4. The method according to claim 1, wherein the distance between the tip of the electrospinning needle and the surface of the receiving solution is 4 to 10cm, and the tip of the electrospinning needle is connected to a high voltage of +10kV to +15 kV.
5. The method according to claim 1, wherein the polymer is any one selected from the group consisting of L-polylactic acid, polycaprolactone, polyglycolic acid, and poly (glycolide-co-lactide).
6. The method of claim 1, wherein the polymer solution is ultrasonically oscillated prior to electrospinning.
7. The method according to claim 1, further comprising immersing the micro fiber having the three-dimensional orientation structure obtained after dropping into the receiving liquid in a pre-gelling solution to gel;
wherein the pre-gel solution is a cell matrix removal pre-gel solution or a biological pre-gel solution; the biological pre-gel solution is selected from any one of a pre-gel solution of gelatin, a pre-gel solution of collagen and a polypeptide pre-gel solution.
8. The method of claim 7, further comprising adding a neurotrophic factor to the pre-gel solution prior to gelation.
9. The method for preparing a nerve graft according to claim 8, wherein the content of the neurotrophic factor in the pre-gel solution is 1ug/mL to 20 mg/mL.
10. The method of claim 8, wherein the neurotrophic factor comprises at least one of a neurotrophic factor, a basic fibroblast neurotrophic factor, a brain-derived neurotrophic factor, a neurotrophic factor 3, and a glial cell-derived neurotrophic factor.
11. The method for preparing the nerve graft according to claim 7, wherein the micro fibers are soaked in the pre-gel solution at a temperature of less than 37 ℃ and then gelled at 37 ℃.
12. The method for preparing the nerve graft according to claim 11, wherein the micro fibers are soaked in the pre-gel solution at 2-10 ℃ and then gelled at 37 ℃.
13. The method of preparing a nerve graft according to claim 7,
the acellular matrix pre-gel solution is a nerve acellular matrix pre-gel solution.
14. The method of claim 7, wherein the acellular matrix pre-gel solution is preferably a porcine-derived peripheral nerve acellular matrix pre-gel solution.
15. The production method according to claim 1, further comprising removing the receiving liquid from the surface of the microfibers having the three-dimensional oriented structure obtained after falling into the receiving liquid.
16. The method of preparing a nerve graft as claimed in claim 15, wherein removing the receiving fluid from the surface of the micro-fibers comprises: washing with ethanol, soaking in pure water, and sucking off water attached to the surface of the micrometer fibers after soaking.
17. A nerve graft having a three-dimensional oriented structure, which is produced by the production method according to any one of claims 1 to 16.
18. A manufacturing device for preparing the nerve graft with the three-dimensional orientation structure according to claim 17, which comprises an electrospinning device and a rotary receiving device, wherein the rotary receiving device comprises a base, a motor and a rotary table, the base is detachably connected with the rotary table, the base is provided with a mounting groove, the motor is arranged in the mounting groove and is coaxially connected with the rotary table for driving the rotary table to rotate, and the motor is provided with a speed controller;
the electrostatic spinning device comprises an injection needle, wherein the injection needle is positioned above the liquid tank and is arranged at an interval with the liquid tank.
19. The manufacturing equipment as claimed in claim 18, wherein the containing cavity is a cylindrical containing cavity, and the diameter of the fixing shaft is 1/4-2/5 of the diameter of the containing cavity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810497604.XA CN108379655B (en) | 2018-05-22 | 2018-05-22 | Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810497604.XA CN108379655B (en) | 2018-05-22 | 2018-05-22 | Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108379655A CN108379655A (en) | 2018-08-10 |
| CN108379655B true CN108379655B (en) | 2020-06-30 |
Family
ID=63071118
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810497604.XA Active CN108379655B (en) | 2018-05-22 | 2018-05-22 | Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108379655B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111214709A (en) * | 2020-03-27 | 2020-06-02 | 中国医科大学 | Novel double-layer nerve conduit and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101664346A (en) * | 2009-09-02 | 2010-03-10 | 南通大学 | Artificial nerve graft prepared by electrostatic spinning and preparation method and special device thereof |
| CN101845675A (en) * | 2010-05-10 | 2010-09-29 | 北京化工大学 | Electrostatic spinning method and device for preparing nano long fibers arranged in single direction along fiber axis |
| CN102358959A (en) * | 2011-08-16 | 2012-02-22 | 中山大学 | Method and device for preparing electrospinning fiber bracket with three-dimensional structure |
| CN102688076A (en) * | 2011-03-25 | 2012-09-26 | 广州迈普再生医学科技有限公司 | Nerve conduit and preparation method thereof |
| CN102281907B (en) * | 2008-08-11 | 2014-06-25 | 菲布拉利恩公司 | Biocomposites and methods of making the same |
-
2018
- 2018-05-22 CN CN201810497604.XA patent/CN108379655B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102281907B (en) * | 2008-08-11 | 2014-06-25 | 菲布拉利恩公司 | Biocomposites and methods of making the same |
| CN101664346A (en) * | 2009-09-02 | 2010-03-10 | 南通大学 | Artificial nerve graft prepared by electrostatic spinning and preparation method and special device thereof |
| CN101845675A (en) * | 2010-05-10 | 2010-09-29 | 北京化工大学 | Electrostatic spinning method and device for preparing nano long fibers arranged in single direction along fiber axis |
| CN102688076A (en) * | 2011-03-25 | 2012-09-26 | 广州迈普再生医学科技有限公司 | Nerve conduit and preparation method thereof |
| CN102358959A (en) * | 2011-08-16 | 2012-02-22 | 中山大学 | Method and device for preparing electrospinning fiber bracket with three-dimensional structure |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108379655A (en) | 2018-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220162412A1 (en) | Three-dimensional porous hybrid scaffold and manufacture thereof | |
| CN109880151A (en) | A kind of preparation method and porous support materials of hydrogel porous microsphere | |
| JP5855151B2 (en) | Silk biomaterial and method of using the same | |
| Ayres et al. | Nanotechnology in the design of soft tissue scaffolds: innovations in structure and function | |
| CA2621206C (en) | Fibrous 3-dimensional scaffold via electrospinning for tissue regeneration and method for preparing the same | |
| CN101579246B (en) | Artificial silk fibroin nano-fiber nerve repair conduit and preparation method thereof | |
| US20200330644A1 (en) | Methods of forming three-dimensional tissues scaffolds using biological fiber inks and methods of use thereof | |
| US20060095137A1 (en) | Nanofibrous nonwoven membrane of silk fibroin for guided bone tissue regeneration and manufacturing method thereof | |
| US20130312638A1 (en) | Systems, devices and methods for the fabrication of polymeric fibers | |
| US9683011B2 (en) | Controlled cross-linking processing of proteins | |
| WO2003024297A2 (en) | Cell storage and delivery system | |
| WO2011026323A1 (en) | Artificial neural implant prepared by electrostatic spinning, preparation method and special device therefor | |
| CN116966345A (en) | 3D printable biogel and method of use thereof | |
| CN102218160A (en) | Preparation and application of nerve tissue matrix derived tissue engineering scaffold material | |
| KR100751547B1 (en) | Scaffold and method of manufacturing scaffold, and electrospinning device of manufacturing scaffold | |
| CN108379655B (en) | Nerve graft with three-dimensional orientation structure and preparation method and manufacturing equipment thereof | |
| Randhawa et al. | Manufacturing 3D Biomimetic Tissue: A Strategy Involving the Integration of Electrospun Nanofibers with a 3D‐Printed Framework for Enhanced Tissue Regeneration | |
| CN104189954B (en) | A kind of in-situ solidifying tissue engineering bracket and preparation method thereof | |
| Carter et al. | Engineered Biomimicry: Chapter 7. Bioscaffolds: Fabrication and Performance | |
| Sun et al. | Cell-Incorporated Bioactive Tissue Engineering Scaffolds made by Concurrent Cell Electrospinning and Emulsion Electrospinning | |
| Xu et al. | Electrospun three-dimensional nanofibrous scaffolds based on polycaprolactone for stem cells differentiation and bone regeneration | |
| Sharmin et al. | Regenerative engineering: role of scaffolds, cells, and growth factors | |
| Biazar et al. | Nanotechnology for peripheral nerve regeneration | |
| CN107604468B (en) | Processing method and application of micron-sized biological material fiber | |
| PL244645B1 (en) | Method for preparing an injectable biocompatible carrier of drugs, cells or combinations thereof, in the form of microforms, an injectable composition containing the said carriers, and its use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |